Estradiol-receptor Complex Research Articles

The binding of [ 3H]oestradiol-receptor complex to hypothalamic chromatin of male and female mice

1. 1. Histories and masking acidic proteins were removed from hypothalamic chromatin in order to evaluate/measure the number of available acceptor sites for the [ 3H]oestradiol-receptor complex. This number increases after dehistonizing and unmasking and is lower than published values for comparable preparations. 2. 2. No sex-related difference in [ 3H]oestradiol-receptor binding to hypothalamic chromatin in ritro was observed. 3. 3. Failure to observe such a difference suggests that sexual differentiation and steroid sensitivity cannot be attributed to marked differences in the degree of chromatin masking.

Conversion of estrogen receptor from a state with low affinity for estradiol into a state of higher affinity does not require 4S to 5S dimerization.

This study was undertaken to establish whether the heat-promoted conversion of receptor-estradiol complex (RE2) from a state with fast into a state with slow E2 dissociation requires 8S/4S to 5S transformation. The calf uterine estrogen receptor labeled with [3H]E2 at 0 C (state with low affinity for E2) was immobilized on hydroxylapatite (HAP) in the absence (8S oligomer) or presence (4S monomer) of 0.4 M KCl and heated at 28 C in the presence of unlabeled diethylstilbestrol. Under these conditions, the dissociation of [3H]E2 was biphasic and occurred at rates similar to those obtained with R[3H]E2 free in cytosol. In contrast to the latter, however, the heat-promoted conversion of HAP-immobilized R[3H]E2 into a state of higher affinity for E2 was not accompanied by receptor dimerization, since the HAP eluate (0.4 M phosphate buffer) contained only the 4S monomer; upon reheating or desalting of this eluate, 4S to 5S dimerization occurred. The heat-promoted formation of 4S RE2 monomers with higher affinity for E2 was not due to monomer-HAP interactions, since even after elution from HAP, the 4S RE2 remained in the state of higher affinity for E2. Addition of pyridoxal 5'-phosphate to slow dissociating, high affinity 5S R[3H]E2 dimers free in cytosol induced rapid [3H]E2 dissociation, although the receptor remained unaltered in the transformed dimerized state. The effect of PLP was readily reversed by the addition of lysine. It is proposed that the 4S receptor monomers exist in two conformational states; upon E2 binding to the low affinity state, conformational changes result in stronger interactions between the steroid and the amino acid residues of the estrogen-binding domain; thus, the rate of E2 dissociation decreases. The formation of this 4S RE2 state with higher affinity for E2 is independent from receptor dimerization. A model is presented in which 4S to 5S transformation may lead to stabilization of 4S monomers in the conformation with higher affinity for E2.

Interaction of the antiestrogen [ 3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [ 3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly ( t 1 2 1,3 approx 30 h at 20°C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [ 3H]estradiol-receptor complexes. [ 3H]H1285-Receptor complexes sediment at approx 6S on 5–20% sucrose density gradients containing 0.3 M KC1 with or without 10 mM molybdate. This is in contrast to [ 3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KC1) & Peak II (0.25 M KC1). However, [ 3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KC1, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H 1285 can compete with [ 3H]estradiol for binding to both forms of the estrogen receptor and [ 3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [ 3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [ 3H]1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [ 3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.

Analysis of two forms of molybdate-stabilized estrogen receptor.

We recently reported that the calf uterine estrogen receptor prepared in 10-mM molybdate elutes as two components (peaks I and II) from DEAE-Sephadex columns with a linear KCl gradient. We have extended these studies and report that unoccupied cytosol receptors also elute as two peaks (0.21 and 0.25 m KCl), as determined by postlabeling experiments with [3H]estradiol. Since molybdate-stabilized cytosol estrogen receptors incubated in the presence of 0.3 M KCl as well as estrogen receptors in peaks I and II did not demonstrate receptor binding to DNA-cellulose, we conclude that both receptor forms are nonactivated. Also, [3H]estradiol-receptor complexes from cytosol prepared in 10 mM molybdate sediment as 6.7S in 5-20% sucrose density gradients containing 0.3 M KCl, whereas receptor complexes from cytosol prepared without molybdate sediment as 4.5S. Both peaks I and II were eluted from DEAE-Sephadex columns prepared with either 10 mM molybdate or 10 mM tungstate, with both phosphatase inhibitors having effectively blocked salt activation of the receptor. However, receptor preparations in the presence of 10 mM arsenate were not eluted from DEAE-Sephadex, and arsenate was unable to inhibit receptor activation by KCl. Sucrose density gradient analysis of peaks I and II indicates that peak I sediments at approximately 4.8S, whereas peak II sediments at approximately 6.3S. If pooled fractions of the leading portion of peak I are rechromatographed on another DEAE-Sephadex column, both peaks I and II are recovered. Likewise, if pooled fractions of the descending portion of peak II are rechromatographed, both peaks I and II were measured. Therefore, we conclude that there may be an equilibrium between two forms of the molybdate-stabilized calf uterine estrogen receptor.

Activation of [ 3H]estradiol- and [ 3H]H1285-receptor complexes: Effect of salt versus ATP on molybdate-stabilized estrogen receptors

The activation by salt or ATP of [ 3H]estradiol- and [ 3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [ 3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KC1 caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [ 3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [ 3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resinbinding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [ 3H]estradiol- and [ 3H]H1285-receptor activation suggests that estrogen-receptor complexes are “fully” activated as compared to “partially” activated antiestrogen-receptor complexes.

Estrogen receptor transformation to a high-affinity state without subunit-subunit interactions.

We have tested the ability of monomeric rat estrogen receptor (generated by dissociating the native receptor aggregate with KCl and binding to hydroxylapatite) to undergo heat-induced transformation to a state with altered chromatographic properties and estradiol dissociation kinetics. Participation of nonadsorbed cytosolic components in the transformation process was eliminated by washing the adsorbed receptor prior to incubation at 28-30 degrees C for 30 min. We found that monomeric estradiol-receptor complexes can be converted to a form which coelutes with transformed receptor during hydroxylapatite chromatography and which has a slow estradiol dissociation rate characteristic of transformed receptor. Monomeric receptor does not, however, display cooperative ligand-binding behavior, strongly suggesting adsorbed monomers cannot dimerize or interact with other adsorbed cytosol proteins. Maintenance of the monomer form was further verified by the sedimentation of eluted receptor solely as a 4S species. Although conversion of monomers to the slow-dissociating state was incomplete (congruent to 80%) on hydroxylapatite, eluted receptor showed only a slow dissociation rate component. The data are consistent with a model in which receptor monomers are in equilibrium between low-affinity and high-affinity conformations. Dimerization of the high-affinity receptor may shift the equilibrium to greatly favor the high-affinity (transformed) state.

Binding of [ 3H]estradiol- and [ 3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [ 3H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [ 3H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.

Normalization of estradiol receptor kinetics and hormonal activity in uterus of streptozotocin-induced diabetic rats treated with insulin.

In response to ip 17 beta-estradiol injection, ovariectomized streptozotocin-induced diabetic rats showed a decreased early and late stimulation of uterine protein synthesis as compared to nondiabetic controls. An early nuclear loss of estradiol-receptor complex and an accelerated estradiol clearance from the circulating blood were also observed in these animals. To establish whether these alterations in hormone kinetics and activity were related to the diabetic state, further investigation was done by treating diabetic animals with insulin, during 18 consecutive days after 3 to 8 months of long standing diabetes (type I treatment) or during 10 weeks starting immediately at the onset of the streptozotocin-induced disease (type II treatment). Both types of treatment either restored (type I) or preserved (type II) a normal body weight, low glycemia, and absence of glycosuria. Both types of treatment also restored a normal hormonal clearance so that higher plasma levels of estradiol at 30 and 90 min were noted after ip injection when compared to untreated diabetics. Nuclear retention of the estradiol-receptor complex was significantly higher in treated diabetics at 210 min as compared to the nondiabetic controls. Finally, stimulation of early protein synthesis at 90 min was normalized after both types of treatment. From the above observations it is concluded that insulin treatment restored a normal estrogen metabolism in diabetics, resulting in a prolonged retention of the estradiol-receptor complex in the uterine nuclei; the latter event in turn elicited a normal hormonal activity on protein synthesis. A direct influence of the diabetic state and of insulin treatment on the receptor kinetics and/or on the tissue response to the hormonal stimulus may however not be excluded.

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with nonidet P-40

The extraction of [ 3H]estradiol- and [ 3H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [ 3H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [ 3H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [ 3H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S. The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [ 3H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.

Influence of intratumoral estradiol biosynthesis on estrogen receptors.

According to the common theory on steroid receptor mechanism, the amount of cytosol estrogen receptor, as well as the intranuclear estradiol-receptor complex, is influenced by intracellular estradiol levels [6]. Intracellular estradiol arises from two sources: from peripheral estradiol by transport into the tumor cell and by intratumoral biosynthesis from other steroids. It is well known that cytosol receptor values are invariably low in tumors from women with high (premenopausal) serum estrogens, probably owing to blockade of the receptor by endogenous estradiol. In such cases intratumoral estradiol formation will probably be less important. On the other hand, receptor-rich as well as receptor-poor tumors are found in women with low (postmenopausal) peripheral estrogens, and in such cases intratumoral estradiol biosynthesis may be of clinical importance. The following discussion will be restricted to the situation present in the postmenopausal woman and will cover three pathways of intratumoral estradiol formation: (a) reduction of estrone; (b) ring A aromatization of androgens; and (c) hydrolysis of estrogen conjugates.

Protease inhibitor antipain reduces nuclear binding of estrogen-receptor complex in MCF-7 breast tumor cells.

We have initiated studies to ascertain the effects of protease inhibitors on specific steroid hormonal-related events as they occur in cultured cells, in an attempt to identify hormone-related processes perturbed by these inhibitors. We report here that the presence of antipain during incubation of cultured MCF-7 breast tumor cells with estradiol-containing medium considerably reduces the extent of nuclear binding of the estradiol-receptor complex.

The role of arginyl residues in estrogen receptor activation and transformation.

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Estradiol and tamoxifen interaction at receptor sites at 37 C.

Mature rat uterine cytosol was pretreated with dextran-coated charcoal at 0 C for 2 h. This renders the subsequently formed estradiol-receptor complex thermostable at 37 C and also uncovers antiestrogen binding sites, possibly by removing an endogenous ligand. A sharp distinction is found between tamoxifen and estradiol as receptor ligands in pretreated cytosols in that tamoxifen will inhibit estradiol binding, on incubation at 37 C, only if dithiothreitol (DTT) had been included in the pretreatment solution. The presence of tamoxifen as the sole ligand in cytosol pretreated in the presence of DTT does not protect the estradiol receptor from thermal inactivation and following 37 C incubation tamoxifen is found bound exclusively to antiestrogen binding sites. Incubating the cytosol at 37 C with an equimolar mixture of estradiol and tamoxifen results in a very large increase in receptor-bound estradiol. This effect is attributed to the presence of tamoxifen complexed with antiestrogen sites. Tamoxifen in such equimolar ligand mixture binds to antiestrogen sites and is excluded from receptor sites by the estradiol, whose affinity for these sites is much greater than that of tamoxifen.

Interactions of nuclear estrogen receptor with DNA and RNA

The interactions of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNA-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U 1G 1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.

Antiestrogen pharmacology and mechanism of action

Antiestrogens are nonsteroidal compounds that are capable of antagonizing many of the actions of estrogens. They have long half-lives in serum, and during their action in vivo, the antiestrogens C1628, U23,469 and tamoxifen undergo bioactivation to more polar forms that have a higher affinity for the estrogen receptor. Utilizing the high affinity antiestrogens C1628M and trans-hydroxytamoxifen in radiolabeled form, we find differences in the interaction of the estradiol receptor complex and antiestrogen receptor complex with DNA cellulose and differences in their association in the nucleus. In MCF-7 human breast cancer cells, there are marked differences in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with antiestrogen (5.5 +/- 0.06 s, 5.93 +/- 0.20 nm Stokes radius, 137,000 mol. wt) as opposed to estrogen (4.1 +/- 0.03 s, 4.84 +/- 0.20 nm Stokes radius, 83,000 mol. wt). Our data are consistent with the association of the antiestrogen receptor with an additional nuclear protein of approx. 55,000 mol. wt. This antiestrogen promoted change in receptor association with another cellular component, which may function as a possible nuclear "blocking protein" and the alterations in antiestrogen-receptor association with DNA and nuclei, may be important aspects of the estrogen antagonist and growth inhibiting properties of these compounds. In addition to binding directly to the estrogen receptor, antiestrogens can be found associated with binding sites that are distinct from the estrogen receptor. These sites are present in a wide variety of estrogen target and non-target tissues in the rat, and in estrogen receptor positive and negative human breast cancer cell lines (MCF-7, T47D, MDA-MD-231) that differ markedly in their sensitivity to antiestrogens. These sites have a subcellular distribution and ligand binding specificity very different from those of the estrogen receptor. While their wide distribution and order of binding affinities for different antiestrogens raise questions about their role in mediating directly the classically recognized estrogen antagonism of antiestrogens, these sites might alter the apparent distribution volume of antiestrogens and hence their biological potency and pharmacokinetics.

Estrogen and antiestrogen binding to different forms of the molybdate-stabilized estrogen receptor.

We report that the calf uterine estrogen receptor, prepared in a Tris-molybdate buffer, bound by 10 nM [3H]estradiol and eluted by a KCl gradient from DEAE-cellulose columns, yielded only one very sharp receptor peak. Estrogen receptor prepared in phosphate buffer with molybdate and eluted with KCl also yielded only one sharp peak on DEAE-cellulose. However, if DEAE-Sephadex (with phosphate buffer plus molybdate) was used, the [3H]estradiol-receptor complex eluted with two sharp peaks at approximately 0.21 and 0.25 M KCl (Peaks I and II, respectively). But the high-affinity antiestrogen, [3H]H1285, bound to estrogen receptor, eluted only as Peak I and not as Peak II. Both the [3H]estradiol and [3H]H1285 binding peaks were saturable since they could be eliminated with 200-fold excess estradiol. Therefore, ion exchange chromatography using different resins and/or buffers may be useful for determining physicochemical differences in estrogen versus antiestrogen receptor complexes.

Acceptor sites for the oestrogen receptor in hen oviduct chromatin

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

Sex-specific alterations in estrogen receptor interactions following induced androgen imbalance in vivo

Androgens have been shown, under in vitro conditions, to be capable of impeding the rate of formation of estrogen-receptor complexes in target tissues of the rat. The present study was designed to investigate effects of abnormal androgen levels in vivo on various estrogen receptor systems. Serum levels of testosterone (T) and 5α-DHT were measured in adult neonatally-androgenized rats. The T/DHT ratio in the androgenized animal was 0.70, compared to 4.37 in the normal adult rat, and this was unaccompanied by any change in the sum of the 2 androgens. Estradiol levels were equivalent to those of normal rats in estrus. In addition to this animal model, castrate rats of both sexes which had been administered chronic high dosages of various androgens were examined. Equilibrium binding studies of cytosol from uterus, anterior putuitary and hypothalamus showed that estrogen receptors were not modified in either of these animal models, with specific reference to affinity of estradiol binding or concentration of binding sites. The association rate kinetics for estradiol-receptor complex formation in uterus and pituitary were unaffected by androgen administration to ovariectomized animals; however, in the corresponding male castrate model, interaction between estradiol and its pituitary cytosol receptor was accelerated by in vivo exposure to androgens, and the effect was dependent on the nature and level of the androgen used. Neonatally-androgenized rats also manifested an initial rate of estradiol-receptor interaction which was appreciably higher than control values. The reversibility of the androgen effect on the estrogen receptor was demonstrated in an in vitro protocol. The results indicate that the in vivo effects of androgens on estrogen receptor kinetics are sex-dependent and, where observed, any influence was of a stimulatory nature. Moreover, it appears that the nature of the androgen present in vivo is at least as important a determinant as the total androgen concentration, and that androgens do not engender permanent changes in the ability of the estrogen receptor to interact with estradiol.